In animals, differentiation of germline from soma usually takes place during embryogenesis. Genes and their products that are preferentially expressed in the embryonic germ cells are regarded as candidates for maintaining germline fate or promoting germline identity. In Drosophila, for example, the protein encoded by the germline gene vasa is specifically restricted to the germ cells, while products of the gap gene hunchback (hb), a somatic gene, are preferentially expressed in the neuroblasts. In this study, we report the expression of both messenger RNA and protein encoded by Aphb, an hb orthologue in the asexual viviparous pea aphid Acyrthosiphon pisum, in germ cells as well as in neuroblasts. We infer that expression of Aphb messenger RNA in the germ cells during the formation of germaria is required for the anterior localization of Aphb in the protruding oocytes. Germarial expression and anterior localization of ApKrüppel was also identified but, unlike Aphb, its expression was not detected in the migrating germ cells. Very similar patterns of hb expression were also identified in the green peach aphid Myzus persicae, suggesting that germline expression of hb is conserved within the Aphididae. To date, this pattern of hb germline expression has not been reported in other insects.

RNA in situ hybridization (ISH), including chromogenic ISH (CISH) and fluorescent ISH (FISH), has become a powerful tool for revealing the spatial distribution of gene transcripts in model organisms. Previously, we developed a robust protocol for whole-mount RNA CISH in the pea aphid Acyrthosiphon pisum, an emerging insect genomic model. In order to improve the resolving capacity of gene detection, we comprehensively surveyed current protocols of whole-mount RNA-FISH and developed protocols that allow, using confocal microscopy, clearer visualization of target messenger RNAs (mRNAs) - including those subcellularly localized and those with spatially overlapping expression. We find that Fast dye-based substrate fluorescence (SF), tyramide signal amplification (TSA), and TSA Plus all enable identifying gene expression thanks to multiplex amplification of fluorescent signals. By contrast, methods of direct fluorescence (DF) do not allow visualizing signals. Detection of a single gene target was achieved with SF and TSA Plus for most mRNAs, whereas TSA only allowed visualization of abundant transcripts such as Apvas1 and Appiwi2 in the germ cells. For detection of multiple gene targets using double FISH, we recommend: (i) TSA/TSA, rather than TSA Plus/TSA Plus for colocalized mRNAs abundantly expressed in germ cells, as proteinase K treatment can be omitted; and (ii) SF/TSA Plus for other gene targets such as Apen1 and Apen2 as inactivation of enzyme conjugates is not required. SF/SF is not ideal for double FISH experiments due to signal blurring. Based on these new conditions for RNA-FISH, we have obtained a better understanding of germline specification and embryonic segmentation in the pea aphid. We anticipate that the RNA-FISH protocols for the pea aphid may also be used for other aphids and possibly other insect species, thus expanding the range of species from which useful insights into development and evolution may be obtained.

Aphids are hemimetabolous insects that undergo incomplete metamorphosis without pupation. The annual life cycle of most aphids includes both an asexual (viviparous) and a sexual (oviparous) phase. Sexual reproduction only occurs once per year and is followed by many generations of asexual reproduction, during which aphids propagate exponentially with telescopic development. Here, we discuss the potential links between viviparous embryogenesis and derived developmental features in the pea aphid Acyrthosiphon pisum, particularly focusing on germline specification and axis determination, both of which are key events of early development in insects. We also discuss potential evolutionary paths through which both viviparous and oviparous females might have come to utilize maternal germ plasm to drive germline specification. This developmental strategy, as defined by germline markers, has not been reported in other hemimetabolous insects. In viviparous females, furthermore, we discuss whether molecules that in other insects characterize germ plasm, like Vasa, also participate in posterior determination and how the anterior localization of the hunchback orthologue Ap-hb establishes the anterior-posterior axis. We propose that the linked chain of developing oocytes and embryos within each ovariole and the special morphology of early embryos might have driven the formation of evolutionary novelties in germline specification and axis determination in the viviparous aphids. Moreover, based upon the finding that the endosymbiont Buchnera aphidicola is closely associated with germ cells throughout embryogenesis, we propose presumptive roles for B. aphidicola in aphid development, discussing how it might regulate germline migration in both reproductive modes of pea aphids. In summary, we expect that this review will shed light on viviparous as well as oviparous development in aphids.

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